Methods for obtaining from Chlorella extract polysaccharides having immunomodulating properties

ABSTRACT

Chlorella  extracts containing high molecular weight  Chlorella  polysaccharide and polysaccharide complexes show immune modulatory, specifically immune stimulatory, activity. The polysaccharide and polysaccharide complexes contain glucose and any combination of: galactose, rhamnose, mannose and arabinose, as well as N-acetyl glucosamine and N-acetyl galactosamine. The extracts may be treated with pronase, DNAse, RNAse and proteases to remove unassociated nucleic acids, ribonucleic acids and proteins. The extracts may also undergo treatment to effect cleavage of specific glycosidic linkages, the linkages being defined by their susceptibility to cleavage by amylase, amyloglucosidase, cellulase or neuraminidase.  Chlorella  extracts may be administered to a mammal to increase proliferation of splenocytes and increase production of cytokines such as IL-6, IL-10, INF-γ and TNF-α. They may be used as a supplement to a vaccination regimen.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 09/925,953, now U.S. Pat. No. 6,551,596, which was filed on Aug. 10,2001 and which claims the benefit of U.S. Provisional Application No.60/224,014, filed Aug. 10, 2000, the contents of which are hereinincorporated by reference.

FIELD OF INVENTION

The present invention relates to Chlorella extracts for use asimmunomodulators.

BACKGROUND OF THE INVENTION

Chlorella is a unicellular green algae that has been called asun-powered supernutrient. Attested beneficial properties of this ediblemicroalgae include wound healing, detoxification, constipation reliefand growth stimulation. A number of studies have also indicated thatChlorella has beneficial effects on the immune system, both in vitro andin vivo.

Chlorella occurs in both fresh water and marine water. Species of theChlorella genus exhibit striking diversity of physiological andbiochemical properties (Kessler, E. “Phycotalk” 1989, 1:141-153; V.Rastogi Publ., New Delhi, India). Chlorella produces little celluloseand other indigestible cell wall material, and hence has beenextensively investigated as a possible new source of food, especially asfeedstock (Lee, Robert E. “Phycology” 2^(nd) edition; 1989, page 281;Cambridge University Press).

Chlorella has the highest content of chlorophyll of any known plant. Italso contains vitamins, minerals, dietary fibre, nucleic acids, aminoacids, enzymes, etc. It contains more than 9% fats (out of whichpolyunsaturated fatty acids [PUFA] represent about 82%). The vitamincontent consists of provitamin A, vitamins B₁, B₂, B₆, niacin, B₁₂,biotin, vitamin C, vitamin K, pantothenic acid, folic acid, choline,lipoic acid, ionositol, PABA etc. Among the minerals present the mostimportant are P, K, Mg, S, Fe, Ca, Mn, Cu, Zn and Co. The maincomponents of Chlorella cells are about 60% protein (composed of allbasic amino acids), and 20% carbohydrate.

Aqueous extracts of Chlorella pyreinodosa has been used for itsnutritive content as well as for other health benefits. Studies reporton numerous health benefits including improved immune system functionand detoxification of harmful toxins. It was introduced as a health foodin the USA in 1977 when novel technology processes were developed whichmade it more digestible and has been the largest selling health foodsupplement in Japan for a number of years. The Taiwan Chlorella companyis the world's largest supplier of Chlorella, and sells the productworldwide to Asia, Europe and North America, under the following brandnames: Algea, Bio-REU-RELLA, Green Gem, Green Boost, Green Nature, GreenPower, Joyau Vert and Natural Boost.

A number of studies have documented C. vulgaris extracts which haveanti-tumor activity, as well as activity against Listeria and E. coli(Tanaka et al. Immunopharmacol. Immunotoxicol., 1990, 12(2):277-291;Tanaka et al. Cancer Immunol. Immunother., 1998, 45(6):313-320; Hasegawaet al. Int. J. Immunopharmacol., 1990, 12(8):883-891). These activitiesappear to be immune-mediated rather than direct toxicity against thetumor or pathogen.

A number of Chlorella extracts are available commercially, includingproducts by Swiss Herbal and Nature's Way. The Swiss Herbal product isidentified as pure Chlorella broken cells containing Protein 61%,Carbohydrate 21.1%, Fat 11.0%, Chlorophyll 2.866%, RNA 2.94% and DNA0.28%.

Other publications related to the present field include the following:

Japanese patent application laid open No. Sho 58-15920 disclosespolysaccharides from fresh water Chlorella having immune potentiator andanti-tumor activity.

Neveu et al. Experientia, 1978, 34(12):1644-1645 discloses that C.pyrenoidosa is an immune response modulator.

Vermeil and Morin CR Seances Soc. Biol. Fil., 1976, 170(3):646-649discloses that C. pyrenoidosa, presumably by nature of its cell wall,protects mice against sarcoma grafting.

Miyazawa et al. J. Ethnopharmacol., 1988, 24(2-3):135-146 discloses thatC. pyrenoidosa cells or extract mediate host immune enhancement of theanti-tumor response.

Umezawa et al. Chemotherapy, 1982, 30(9):1041-1046 and Komiyama et al.Chemotherapy, 1986, 34:302-307 disclose that the acidic polysaccharideChlon A from C. pyrenoidosa has immune enhancing and anti-tumor effects.Chlon A contains rhamnose, arabinose, glucose, galactose and glucuronicacid.

White and Barber Biochimica Biophysica Acta, 1972, 264:117-128 disclosesan 88 kDa acidic polysaccharide from C. pyrenoidosa containing mainlyrhamnose, as well as arabinose, galactose, xylose, mannose andglucuronic acid.

U.S. Pat. No. 4,533,548 discloses acidic polysaccharide CH-1 from C.pyrenoidosa containing mainly rhamnose, as well as arabinose, galactose,glucose and glucuronic acid. The polysaccharide was obtained via gelfiltration with Sephadex G-75.

U.S. Pat. No. 4,831,020 discloses a polysaccharide extract from C.minutissima, a marine Chlorella, with immune-stimulating and anti-tumoractivity. This patent states that polysaccharides from marine Chlorellaspecies are more effective in activating immunity than fresh waterChlorella species. The polysaccharide extract was obtained via gelfiltration with Sephadex G-50.

U.S. Pat. No. 4,786,496 discloses a lipid and glycolipid fraction ofmarine Chlorella with immuno-potentiating activity.

Kojima et al. J. Retic. Soc., 1973, 14:192-208 discloses a 1,250-1400 Dareticuloendothelial system (RES)active glucan from Chlorella.

U.S. Pat. No. 3,462,412 discloses a process for preparing aRES-stimulating extract from Chlorella.

Japanese patent application, publication no. 06248003 discloses aChlorella extract of 15 to 25 kDa, comprising polysaccharides containingpredominantly galactose, with anti-neoplastic activity.

Mizuno et al. Bull. Fac. Agr. Shizuoka Univ. (Shizuoka Daigaku NogakubuKenkyu Hokoku), 1980, 30:51-59 discloses two fractions of neutralglycans from Chlorella, both apparently of small molecular weight.

Ukai et al. Ann. Proc. Gifu Pharm. Univ. (Gifu Yakka Daigaku Kiyo),1990, 39:44-48 discloses two polysaccharides, CP-I and CP-II, from C.pyrenoidosa with RES-stimulating activity. CP-I comprises glucose,fucose, rhammose, galactose and mannose; CP-II comprises glucose,galactose, rhamnose and mannose.

Chu et al. Aquaculture, 1982, 29(3-4):241-252 discloses that thepolysaccharide, ethanol-precipitable fraction of five algal speciesincluding Chlorella contains principally glucose, mannose,ribose/xylose, rhamnose and fucose.

SUMMARY OF THE INVENTION

Chlorella extracts prepared according to the invention show immunestimulatory activity in pharmacological and clinical tests. In oneaspect, the extracts provided by the present invention have a higherimmune stimulatory activity than extracts prepared and used in the art.

In one aspect, the invention provides preparations comprising highmolecular weight Chlorella polysaccharide and polysaccharide complexes.The high molecular weight polysaccharide and polysaccharide complexesare about 1×10⁵ Da to about 1×10⁷ Da and constitute at least 22% (w/w)of the total Chlorella-derived content of the extract. In a preferredembodiment, the extract is derived from Chlorella pyrenoidosa.

The high molecular weight polysaccharide and polysaccharide complexesmay be of a selected range, e.g. about 1×10⁵ Da to about 3×10⁵ Da, about3×10⁵ Da to about 5×10⁵ Da, about 5×10⁵ Da to about 6×10⁵ Da, about6×10⁵ Da to about 7×10⁵ Da, about 7×10⁵ Da to about 8×10⁵ Da, about8×10⁵ Da to about 9×10⁵ Da, about 9×10⁵ Da to about 1×10⁶ Da, about1×10⁶ Da to about 2×10⁶ Da, about 2×10⁶ Da to about 3×10⁶ Da, about3×10⁶ Da to about 4×10⁶ Da, about 4×10⁶ Da to about 5×10⁶ Da, about5×10⁶ Da to about 7×10⁶ Da, about 7×10⁶ Da to about 9×10⁶ Da, and about9×10⁶ Da to about 1×10⁷ Da.

The extracts may contain various different percentages of polysaccharideand polysaccharide complexes as a fraction of the totalChlorella-derived content of the extract. The percentage may be at least24% (w/w), at least 26% (w/w), at least 28% (w/w), at least 30% (w/w),at least 35% (w/w), at least 40% (w/w), at least 45% (w/w), at least 50%(w/w), or at least 60% (w/w).

In another aspect, the high molecular weight polysaccharide andpolysaccharide complexes contain glucose and any combination of:galactose, rhamnose, mannose and arabinose.

In another aspect, the high molecular weight polysaccharide andpolysaccharide complexes is substantially free of ribose, nucleic acids,ribonucleic acids and unassociated proteins. The high molecular weightpolysaccharide and polysaccharide complexes may also contain N-acetylglucosamine and N-acetyl galactosamine.

In another aspect, the extracts of the invention retain immunomodulatingactivity upon treatment to remove unassociated DNA, RNA and proteins.Such treatment includes digestion by pronase, DNAse, RNAse andproteases.

In another aspect, the extracts of the invention retain immunomodulatingactivity upon treatment to effect cleavage of specific glycosidiclinkages, the linkages being defined by their susceptibility to cleavageby amylase, amyloglucosidase, cellulase or neuraminidase. Suchsusceptible linkages are typically:

-   -   (i) three or more α-1,4-linked D-glucose units;    -   (ii) α-1,4-linked glucosides;    -   (iii) α-1,4-linked galactosides; or    -   (iv) α-1,4-linked D-glucose.

The invention also provides nutritional compositions containing the highmolecular weight Chlorella extract with at least one energy source whichmay be carbohydrates, fats or nitrogen.

The extracts of the invention may also be used in combination or inmixture with a conventional supplement such as vitamin E, vitamin C andfolic acid. The extracts may also be used with other nutraceuticals suchas fish oils, spirulina and echinacea, especially those nutraceuticalswhich have immunostimulant activity.

The invention also provides a process for obtaining Chlorellapreparations having immunomodulating activity. The process contains thesteps of:

-   -   (a) size fractionating an aqueous extract of Chlorella, and    -   (b) selecting fractions comprising high molecular weight        polysaccharide and polysaccharide complexes of about 1×10⁵ Da to        about 1×10⁷ Da.

The process for obtaining the Chlorella extract may further include thestep of pooling and concentrating the selected fractions. Sizefractionation may be achieved by chromatography, ultrafiltration orultracentrifugation.

The invention also provides a method for modulating the immune responseof a mammal including humans by administering to the mammal an effectiveamount of the high molecular weight Chlorella extract. Such modulationincludes increased proliferation of splenocytes and increased productionof cytokines such as IL-6, IL-10, INF-γ and TNF-α, and may beadvantageously used to treat or prevent bacterial or fungal infections.

The extract may further be administered as a supplement to a vaccinationregimen to further stimulate the immune response. A flu vaccine may beadvantageously used with the extract. The extract may be present as anadjuvant to the vaccines, especially as an oral vaccine adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention in its various embodiments will now be described withreference to the attached drawings.

FIG. 1: Combination size-exclusion chromatography (SEC)/refractive indexdetection (RI)/multi-angle laser light scattering (MALS) chromatogram ofimmune booster preparations IBP-1 or IBP-2. The top trace is the RIchromatogram; the bottom trace is the MALS response;

FIG. 2: Cumulative molecular weight profile of IBP-1;

FIG. 3: SEC chromatogram of IBP-2 using Sephacryl S 1000 SF;

FIG. 4: SEC chromatogram of IBP-2 (or IBP-1) using Sephacryl S 300 HR;

FIG. 5: Stimulation of splenocyte proliferation by various Chlorellaextract fractions resolved by Sephacryl S 300 HR; IBP-6s is the peakshoulder fraction of IBP-6; ‘Cells’ and ‘Cells+DMSO’ are negativecontrols; LPS is lipopolysaccharide, used as a positive control;

FIG. 6: Stimulation of splenocyte proliferation by various Chlorellaextract fractions resolved by Sephadex G100 chromatography of IBP-2;‘Cells’ is a negative control; LPS is a positive control;

FIG. 7: Splenocyte proliferation—Titration curve of IBP-2; ‘Cells’ is anegative control; LPS is a positive control;

FIG. 8: Proliferation assay measuring ³H-incorporation by isolated B- orT-cells in the presence of 25 μg/mL IBP-2;

FIG. 9: Nitric oxide production by BALB/c inflammatory peritonealmacrophages cultured in the presence of various concentrations of IBP-2;‘IFN+LPS’ is a positive control;

FIG. 10: IL-6 production by BALB/c mouse spleen cells in the presence ofvarious concentrations of IBP-2; ‘Con A’ is Concanavalin A; ‘Cells+ConA’and ‘Cells+LPS’ are positive control samples, Concanavalin A is at 10μg/mL; LPS is at 20 μg/mL;

FIG. 11: Effect of IBP-2 compared to 4 mg echinacea (Echin.) on Listeriamonocytogenes proliferation in mice;

FIG. 12: Effect of IBP-2 compared to crude algae and echinacea onCandida albicans proliferation in mice;

FIG. 13: Mouse splenocyte proliferation as measured by ³H-incorporation,cultured in the presence of IBP-2 or commercial Chlorella extracts fromSwiss Herbal and Nature's Way.

FIG. 14: Capillary electrophoresis chromatogram of the dialyzed crudeextract IBP-2. The monosaccharides are assigned to the peaks as follows:ribose at position 9.05; rhamnose at position 10.47; mannose at position10.68; galactose at position 11.14; and glucose at position 11.65.

FIG. 15: Capillary electrophoresis chromatogram of the retained portionafter IBP-2 was passed through a 1MDa MWCO ultrafiltration membrane. Themonosaccharides are assigned to the peaks as follows: N-acetylgalactosamine (GalNAc)at position 7.30; N-acetyl glucosamine (GlcNAc) atposition 7.43; arabinose at position 10.52; rhamnose at position 10.90;mannose at position 11.11; (note that the peak at position 11.01 is abubble; the mannose peak is just to the right of this peak); galactoseat position 11.62; glucose at position 12.13.

FIG. 16: DEAE Sepharose Fast Flow of IBP-2. A sample was applied onPharmacia 1.0×30 cm column and eluted with piperazine/HCl (0.02 M, pH8.8) buffer at a rate of 5 mL/min. NaCl gradient was employed: 0-20% in20 column volumes, then 20-100% in 2 column volumes.

FIG. 17: DEAE Sepharose Fast Flow of retention portion after IBP-2 waspassed through an ultrafiltration membrane with MWCO 1 MDa. A sample wasapplied on Pharmacia 1.0×30 cm column and eluted with piperazine/HCl(0.02 M, pH 8.8) buffer at a rate of 5 mL/min. NaCl gradient wasemployed: 0-20% in 20 column volumes, then 20-100% in 2 column volumes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for enhancing immunity defencemechanisms of mammals including humans. Immunity defences are enhancedby the administration of Chlorella-derived immunomodulators in the formof Chlorella-derived extracts of molecular weights between 1×10⁵ Da and1×10⁷ Da.

A. Chlorella

Species of the Chlorella genus used in the invention includes thefollowing: minutissima, marina, salina, pyrenoidosa, vulgaris, anitrata,antarctica, autotrophica, regularis, among others (see World Catalog ofAlgae, 2^(nd) Edition, pages 58-74; Miyachi et al. (Eds); 1989; JapanScientific Societies Press, the content of which is herein incorporatedby reference).

Mutant strains of Chlorella, either naturally occurring or artificiallyproduced, for example by irradiation (e.g. ultraviolet, X-ray), chemicalmutagens or by site-directed mutagenesis, are within the scope of theinvention. In one embodiment, C. pyrenoidosa and its variants arepreferred. In another embodiment, C. ellipsoidea and its variants arepreferred. Cultivation of Chlorella is carried out by methods known inthe art using suitable media and culture conditions (see for example,White and Barber, Biochimica Biophysica Acta., 1972, 264:117-128).Polysaccharide production may be influenced by physiological andmetabolic manipulation. Composition of the growth media may influencegrowth rate leading to changes in cell wall thickness. Genes responsiblefor growth may be up- or down-regulated. For a method used to transformeukaryotic algae, see for example U.S. Pat. No. 6,027,900; for methodsto select algal mutants, see for example U.S. Pat. No. 5,871,952. Thus,by selection under various conditions, variants of biopolymerimmunomodulators from Chlorella may be manufactured.

B. Chlorella Extracts and Their Preparation

(a) Preparation of Crude Extract:

Crude aqueous extracts of Chlorella are prepared by methods known in theart, including hot water extraction of cultured cells or spray driedcells (U.S. Pat. No. 4,831,020 and U.S. Pat. No. 5,780,096) and solventextraction methods (White and Barber, Biophys. Biochim. Acta, 1972,264:117-128; U.S. Pat. No. 3,462,412). Crude extracts may be obtainedfrom the Taiwan Chlorella company(www.taiwanchlorella.com/product-3.htm).

In one embodiment, the crude extract is prepared from spray-driedChlorella cells with an average moisture content of 0.3%; (the moisturecontent was determined after drying the spray-dried material for 16 hunder a vacuum of 5 mm). The crude extract is prepared by treating thecells with aqueous media, preferably water or weak solutions of organicacids, such as acetic acid, ascorbic acid, benzoic acid, citric acid,lactic acid, maleic acid, propionioc acid, sorbic acid, succinic acidetc., preferably benzoic acid, under gentle agitation. The extractionprocess could be executed at various temperatures ranging from 0 to 100°C., preferably between 50 and 90° C. The yields and immunoactivitycorrelating with chromatographic profiles indicate that 1 h at 80° C. isa suitable combination of time and temperature to perform this stepefficiently.

The residual cells and the cell debris were separated by centrifugationwith a relative centrifugal force (RCF) of 150 to 10,000 g, preferably4,000 to 10,000 g. The time necessary to complete this step is inrelation to the centrifugal force; 20 min. is sufficient at 10,000 g.The supernatant was then micro-filtered. Alternatively, filtration maybe used to remove whole cells and debris, in which case use of a seriesof filters starting from coarse, through medium and ending withmicro-filtration, is necessary. Cross-flow filtration or vibratingmembrane technology is recommended to reduce fouling. Filtration isparticularly sensitive to temperature and time period required forextraction. Centrifugation is therefore the preferred route.

After centrifugation or filtration, the supernatant (or filtrate) may bedried to obtain IBP-1 products in dry form. The drying was achieved bylyophilization, cold air-flow, or preferably by spray-drying.Alternatively, the volume of the extract could be reduced first (to10-50%, preferably 20%), and then the active materials precipitated fromthe solution with suitable precipitants, preferably ethanol or ammoniumsulfate.

IBP-1 was liberated from salts and low molecular mass products. Althougha variety of aqueous media (such as diluted alcohols, various buffers,diluted acetic acid, etc.) could be employed, water was found to be asufficient medium for the dialysis. This step reduced the mass of theextracted material by about 50% and increased its specificimmunoactivity (as judged by effect on stimulation of splenocytes, seebelow) by about 25%. Dialysis could be replaced by desalting with gelfiltration media, such as Sephadex G 25, Bio Gel P 6 or equivalents.Similarly, corresponding ultrafiltration membranes with correspondingmolecular weight cut-offs could be used. After the desalting step, thematerial (IBP-2) may be dried using methods described for IBP-1.

(b) Size Fractionation of Extracts:

Size fractionation of Chlorella extracts can be accomplished by anymethod known in the art, including size exclusion chromatography,sedimentation analysis e.g. gradient centrifugation, andultra-filtration.

IBP-1 or IBP-2 is a mixture of polysaccharides and polysaccharidecomplexes, with average molecular mass of the immunomodulatory fractionof interest ranging from 100 up to 10,000 KDa. Polysaccharide complexesare polysaccharides which are non-covalently associated with anon-polysaccharide biopolymer which, by itself, has no significantimmune activity. Non-polysaccharide biopolymers include DNA and proteinwhich may contribute to the cumulative molecular weight of the extractbut which has no significant immune activity.

In various embodiments, the high molecular weight polysaccharide andpolysaccharide complexes are about 1×10⁵ Da to about 1×10⁵ Da, about3×10⁵ Da to about 5×10⁵ Da, about 5×10⁵ Da to about 6×10⁵ Da, about6×10⁵ Da to about 7×10⁵ Da, about 7×10⁵ Da to about 8×10⁵ Da, about8×10⁵ Da to about 9×10⁵ Da, about 9×10⁵ Da to about 1×10⁶ Da, about1×10⁶ Da to about 2×10⁶ Da, about 2×10⁶ Da to about 3×10⁶ Da, about3×10⁶ Da to about 4×10⁶ Da, about 4×10⁶ Da to about 5×10⁶ Da, about5×10⁶ Da to about 7×10⁶ Da, about 7×10⁶ Da to about 9×10⁶ Da, and about9×10⁶ Da to about 1×10⁷ Da.

Size fractionation to obtain the above fractions is based on principlesof molecular sieving. Typically, size exclusion chromatographytechniques and ultrafiltration methods are employed. The basicprinciples of size exclusion chromatography are well known to those inthe art, and are explained in “Gel filtration: Principles and Methods.Eighth edition., Amersham Pharmacia Biotech AB, Rahhms I Lund, Uppsala,Sweden”. The appropriate columns for fractionating particular ranges canbe readily selected and effectively used to resolve the above fractions,e.g. Sephacryl S 100 HR, Sephacryl S 200 HR, Sephacryl S 300 HR,Sephacryl S 400 HR and Sephacryl S 500 HR or their equivalents. In ananalogous fashion, Sepharose media or their equivalents, e.g. Sepharose6B, 4B, 2B, could be used.

Purification of the polysaccharides or polysaccharide complexes withprotein could be achieved in combination with other chromatographytechniques, including affinity chromatography, ion exchange, hydrophobicinteraction chromatography etc. An example of IBP-2 (the retainate afterIBP-2 was passed through ultrafiltration membrane with MWCO>1 MDa)chromatography using DEAE-Sepharose Fast Flow anion exchangechromatography is given in FIGS. 16 and 17. The figures demmonstratesignificant decrease in the protein content after IBP-2 was passedthrough the ultrafiltration membrane.

Ultrafiltration of the samples could be performed using molecularmembranes with appropriate molecular mass cut-offs. The specificmembranes and procedures used to effect fractionation are widelyavailable to those skilled in the art, as outlined inhttp://www.uku.fi/laitokset/anat/PG/c_method.htm.

In one embodiment, the method used for characterising and quantifyingthese materials is based on combined size exclusion chromatography(SEC)/multi-angle laser light scattering (MALS)/refractive indexdetection (RI). In the hybrid technique (SEC/MALS/RI), an isocratic HPLCexperiment using a Tosohaas GMPWXL SEC column is used to separatemixtures according to molecular size. On-line MALS determines theaverage molecular weight distribution of the eluting biopolymers andhence provides specificity in the analysis. RI detection is used bothfor quantification and to provide the elution profile required inprocessing the MALS data.

An example of the SEC chromatogram obtained for a typical extract isshown in FIG. 1. The top trace is the chromatogram recorded using the RIdetector while the bottom trace is the MALS response at one of thedetectors (90 degrees). The MALS peak is a maximum for the highmolecular mass component which actually corresponds to a smallpercentage of the total extract as can be seen from the upper trace.Thus, although the molecular weight range extends from a few KDa toabout 10 MDa, the weight average molecular weight (MW) for the entireextract is determined to be around 90 kDa. This can be seen most clearlyfrom the cumulative molecular weight profile of IBP-1 (FIG. 2).

IBP-1 or IBP-2 can be further fractionated using suitablechromatographic or ultrafiltration techniques. Size exclusionchromatography matrices with wide fractionation range such as SephacrylS 1000 SF (FIG. 3) resolved the extract into two peaks: the first elutedjust after the void volume; its average molecular mass, as measured byMALS, averaged 1,000 KDa; the second peak eluted just after the firstpeak. The combined fractions representing the first peak were desaltedand dried analogously to that of IBP-1, resulting in IBP-3. Thismaterial was superior in its immunoactivity compared to the second peak(IBP-4). Aqueous media were used in this chromatography procedure,preferably 0.15 M NaCl. Although IBP-1 exhibited higher immunoactivitythan the IBP-2, the difference was insignificant. The contribution tothe immunostimulant activity (increasing gradually with mass) startslevelling off when molecular mass reaches around 500 KDa.

IBP-4 could be further fractionated using suitable chromatographic orultrafiltration techniques. Both ion exchange chromatography (IEC) andSEC were found to be useful for further resolution of IBP-4. SECmatrices with the appropriate fractionation range, for example SephacrylS 300, could be used. Sephacryl S 300 HR resolved IBP-1 or IBP-2 intotwo peaks (FIG. 4). The first peak started eluting in the very lastfractions of the void volume of the column. The majority of the elutedbiopolymers exhibited molecular masses ranging from 100 to 500 KDa. Thecombined fractions representing the first peak were desalted and driedanalogously to that of IBP-1, resulting in IBP-5. This material wassuperior in its immunoactivity to the second peak (IBP-6) which elutedjust after the first peak touched the baseline (FIG. 5). IBP-5represented typically only 30% of the combined mass of both peaks.Aqueous media were used for this chromatography procedure, preferably a0.1M acetate, pH 4.5 buffer with linear NaCl gradient. The difference inthe immunoactivity of IBP-5 and IBP-6 was higher than in that betweenIBP-3 and IBP-4; typically the ratio of CPM_(IBP-3) to CPM_(IBP-4) was5:2, as measured by the level of ³H-incorporation into proliferatingsplenocytes. The process could be, to some extent, simplified by use ofultrafiltration membranes with molecular mass cut-off of about 500 KDa.For instance, Omega ZM 500 membrane successfully could be used.

IBP-6 could be further fractionated using suitable chromatographic orultrafiltration techniques, for instance using chromatographic matricessuch as Sephadex G 100, Sephadex G 75 or analogous media orcorresponding ultrafiltration membranes. However, specificimmunoactivity residing in IBP-6 was significantly weaker than that ofIBP-3 and IBP-5, and therefore was not of prime interest. However,Sephadex G 100 could be efficiently used to remove from IBP-2 themajority of lower molecular weight material (IBP-8), which is associatedwith low immunoactivity (FIG. 6). Typically the ratio of CPM_(IBP-7) toCPM_(IBP-8) was 10:1; (IBP-7 being the high molecular mass andimmunoactive fraction). This purification step could be achieved just aswell with a YM-100 ultrafiltration membrane.

Crude extracts of Chlorella contain about 61% protein and 21%carbohydrate. Processing of Chlorella according to the present inventionresults in a higher percentage of polysaccharide and polysaccharidecomplexes, i.e. the extracts of the invention have a higher percentageof polysaccharide and polysaccharide complexes relative to the totalmaterial derived from Chlorella, compared to a crude extract from brokencells. It is understood that materials unrelated to Chlorella may beadded to the Chlorella extract and that such extracts are within thescope of the invention.

The percentage of polysaccharide and polysaccharide complexes in theextracts of the invention is at least 23% (w/w) of the totalChlorella-derived content of the extract. In various embodiments, thepercentage is at least 24% (w/w), at least 26% (w/w), at least 28%(w/w), at least 30% (w/w), at least 35% (w/w), at least 40% (w/w), atleast 45% (w/w), at least 50% (w/w), or at least 60% (w/w).

The high molecular weight polysaccharide and polysaccharide complexesmay be further purified and isolated to the various percentagesindicated above by removal of non-polysaccharide components. Suchnon-polysaccharide components include DNA, RNA and unassociatedproteins. (Unassociated proteins are defined for the purpose of thepresent application as proteins which are not associated withpolysaccharides in a polysaccharide complex).

One method of removal is the use of digestion enzymes to cleave thenon-polysaccharide components, followed by size fractionation to removethe cleaved products as described in the Examples below (Example 7).Digestion enzymes include pronase, ribonuclease, DNase and proteases, aswell known in the art and described in various text books, one exampleof which is Maniatis et al., Molecular Cloning: A Laboratory Manual(1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Proteases useful for digestion of unassociated proteins include: endo-and exopeptidases, pronase, serine proteases such as trypsin,chymotrypsin and subtilisin, thiol proteases such as papain, andcalcium-requiring proteases such as thermolysin.

Alternatively, non-polysaccharide components may be removed by affinitychromatography, for example by use of DNA- or RNA-binding matrices(Maniatis et al., 1982). Another option is to purify the polysaccharideand polysaccharide complexes away from the contaminating components byuse of polysaccharide binding matrices such as lectins. In anotherembodiment, the extracts of the invention may be treated with glycosidicenzymes under conditions and for a length of time sufficient to effectcleavage of:

-   -   (i) three or more α-1,4-linked D-glucose units;    -   (ii) α-1,4-linked glucosides;    -   (iii) α-1,4-linked galactosides; or    -   (iv) α-1,4-linked D-glucose.

Examples of glycosidic enzymes useful for cleavage of such glycosidiclinkages include: amylase, amyloglucosidase, cellulase andneuraminidase.

C. Characterization of Extracts

Carbohydrate composition, DNA content and amino acid composition of theChlorella extracts of the invention can be determined by any suitablemethod known in the art.

Immune activity of the extracts of the invention are associated withhigh molecular weight Chlorella polysaccharides, defined as thosemacromolecules consisting of monosaccharides joined by glycosidiclinkages. The polysaccharides are present in the extracts in the form offree polysaccharides or complexed polysaccharides (i.e. polysaccharideswhich are non-covalently associated with a non-polysaccharide biopolymerwhich, by itself, has no significant immune activity). In oneembodiment, the protein content of the extract is about 20% to 50%,preferably 20% to 30%. Of this percentage of proteins, about 40% to 60%are associated with polysaccharides.

Non-polysaccharide biopolymers include DNA, protein and possibly RNA,which may contribute to the cumulative molecular weight of the extractbut which has no significant immune activity. Unassociated RNA, DNA andprotein, i.e. those not complexed with the polysaccharides, do notcontribute significantly to immune activity of the extracts. For thepurposes of the present application, unassociated RNA, DNA and proteinare defined functionally as those RNA, DNA and protein which aresusceptible to cleavage by ribonuclease (RNAse), deoxyribonuclease(DNAse) and common proteases of the serine and thiol class. The extractsof the present invention may thus be essentially free or substantiallyfree of unassociated RNA, DNA and protein. By “essentially free” ismeant less than 5% unassociated DNA or RNA and less than 15%unassociated proteins. By “substantially free” is meant less than 2%associated DNA or RNA and less than 10% unassociated proteins.

It is understood that, while the non-polysaccharide biopolymers per selack immune activity, their association with the polysaccharides maycontribute to the immune activity of the polysaccharides since thenon-polysaccharide biopolymers of the complex may fulfill certain stericor polar requirements which enable the polysaccharides to functioneffectively as immunomodulators.

The extracts of the present invention may be digested with amylase,amyloglucosidase, cellulase and neuraminidase without significant lossof immune activity. Immune activity thus apparently resides inpolysaccharides or their complexes which do not contain a substantialamount of three or more α-1,4-linked D-glucose units; α-1,4-linkedglucosides; α-1,4-linked galactosides; or α-1,4-linked D-glucose.However, it is understood that immunomodulatory polysaccharides maycontain the above glycosidic linkages if such linkages are notaccessible to enzyme digestion.

D. Uses of Extracts

Biological response modifiers have been defined as those agents thatmodify the host's biological response by a stimulation of the immunesystem, which may result in various therapeutic effects. One of thecategories of substances belonging to this class is immunomodulators. Asused herein, the term “immunomodulator” refers to an agent which is ableto modulate an immune response. In the context of the present invention,such modulation is an enhancement of the host's immunity defencemechanism.

Chlorella extracts are thought to be primarily a B-cell and macrophagestimulator. One benefit of B-cell immunomodulators is that they canstimulate immune function in those who may have an impaired antibodyresponse to an antigen. Also, a B-cell stimulator might increase therapidity of the antibody immune response when presented with a newinfection. Chlorella extracts provide a safe, efficacious and costeffective alternative for preventative health treatment.

In vitro studies demonstrated that Chlorella extracts stimulatedproliferation of BALB/c mouse spleen cells, and macrophage production ofIL-6 and NO₂ . Chlorella extracts were also examined in vivo, and foundto significantly reduce infection with Listeria monocytogenes, as wellas the fungus Candida albicans (see Examples 8 to 13).

A series of three toxicology trials have been completed for Chlorellaextracts. No effect of Chlorella extract administration was evidentduring the 28-day oral toxicity study in rats. For the acute oraltoxicity in rats, to determine the highest non-lethal or the lowestlethal dose of the product following a single oral administration, thestudy found that the lowest lethal dose of a crude Chlorella extract wasin excess of 2000 mg/kg body weight. The bacterial mutation assay showedthat Chlorella extracts did not exhibit any mutagenic activity under thetest conditions.

A recently completed randomized, double-blind placebo-controlled studyfound that Chlorella extracts demonstrated significant immunostimulatoryeffects in healthy adults receiving the influenza vaccine, compared toplacebo subjects (see Example 14).

In vitro experiments with human blood cells show stimulation ofproduction of interleukins, similar to that seen in the mouse model.

Chlorella extracts of the invention are suitable for use in anycondition or disease state where immune response enhancement ormodulation is desired. In one embodiment, Chlorella extracts may be usedin an effective amount as adjuvants in various forms of mucosal vaccinepreparations, especially for oral administration.

Adjuvants may protect the antigen from rapid dispersal by sequesteringit in a local deposit, or they may contain substances that stimulate thehost to secrete factors that are chemotactic for macrophages and othercomponents of the immune system. Known adjuvants for mucosaladministration include bacterial toxins, e.g., the cholera toxin (CT),the E. coli heat-labile toxin (LT), the Clostridium difficile toxin Aand the pertussis toxin (PT). Chlorella extracts, being an edibleproduct of high molecular weight and themselves immune stimulants, arecandidates for use as adjuvants in oral vaccines.

The term “effective amount” of an immunomodulator refers to an amount ofan immunomodulator sufficient to enhance a host defence mechanism. Thisamount may vary to some degree depending on the mode of administration,but will be in the same general range. If more than one immunomodulatoris used (for example, Chlorella extract in combination with echinacea),each one may be present in these amounts or the total amount may fallwithin this range. The exact effective amount necessary could vary fromsubject to subject, depending on the species, age and general conditionof the subject, the severity of the condition being treated, the mode ofadministration, etc. Thus, it is not possible to specify an exacteffective amount. However, the appropriate effective amount may bedetermined by one of ordinary skill in the art using only routineexperimentation or prior knowledge in the immunomodulator art.

The term “treatment” as used herein covers any treatment of a mammal,particularly a human, and includes:

(i) preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;

(ii) inhibiting the disease, i.e., arresting its development; or

(iii) relieving the disease, i.e., causing regression of the disease.

E. Form of Extracts

The nutritional and pharmaceutical compositions containing Chlorellaextracts of the invention may be formulated and administered in any formsuitable for enteral administration, for example oral administration ortube feeding. The formulations are conveniently administered in the formof an aqueous liquid. The formulations suitable for enteral applicationare accordingly preferably in aqueous form or in powder or granulateform, including tablet form. The powder or granulate may be convenientlyadded to water prior to use. In liquid form, the compositions have asolid content of typically from 0.1% to 50% by weight, preferably from1% to 10% by weight. As a drink, the compositions may be obtained by anymanner known, e.g. by admixing the Chlorella extract with an energysource such as carbohydrates, fats and nitrogen sources.

The nutritional compositions may be in the form of a complete formuladiet (in liquid or powder form), such that when used as sole nutritionsource, essentially all daily caloric, nitrogen, fatty acids, vitamin,mineral and trace element requirements are met. However, the nutritionalcompositions of the invention are preferably intended for use as adietary supplement.

Pharmaceutical compositions of the invention may also be formulated in asingle-dose format, where they comprise Chlorella extracts and apharmaceutically acceptable carrier. Such pharmaceutical compositionsare suitable for enteral administration, such as oral, nasal or rectaladministration. Suitable compositions may be in liquid form or solidform. Dosage of liquid compositions are typically from 0.1% to 50% byweight, preferably from 1% to 10% by weight of Chlorella extract. Dosageof solid compositions are typically from 0.2 mg/kg to 200 mg/kg,preferably from 1 mg/kg to 10 mg/kg of Chlorella extract Thecompositions may be in the form of tablets, hard and soft capsules, andsachets.

Suitable carriers are known in the art. They comprise fillers such assugars or cellulose, binders such as starch, and disintegrators ifrequired.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Preparation of IBP-1

Twenty grams of dried powder of Chlorella pyreinodosa were mixed with100 mL distilled water and heated under gentle stirring for 1 h at 80°C. The material was centrifuged at 10,000 g for 20 min; the residualpellet was washed twice. The combined supernatants were thenmicro-filtered and spray dried. The average yield was 10.5%. Theextraction of the pellet for 1 h at 80° C. could be repeated severaltimes, preferably twice, instead of simple wash.

Alternatively, the combined supernatants were evaporated to ⅕ of theiroriginal volume using a rotary evaporator under reduced pressure, thenprecipitated with ethanol (80% final ethanol content). The mixture waskept at 4° C. for 18 h, then the precipitate was filtered off, washedwith absolute ethanol and vacuum-dried.

EXAMPLE 2 Preparation of IBP-2

The extracted material, prepared as above, was desalted by exhaustivedialysis against water at 4° C. and then spray-dried. The average yieldwas about 7.5%.

EXAMPLE 3 Chromatography of IBP-2 and Preparation of IBP-3

Twenty milligrams of IBP-2 were dissolved in 1 mL of distilled water,pre-filtered with a 0.45 μm filter and loaded on Sephacryl S 1000 HRcolumn (1.0×50 cm) and eluted with 0.15 M NaCl. Chromatography wasmonitored at 280 nm and at 490 nm after interaction of the elutedfractions with a phenol/sulfuric acid reagent. The mixture was resolvedinto two peaks. The major immunoactivity was in the high molecular masspeak (IBP-3). However, the lower molecular mass peak (IBP-4) alsocontained a significant portion of immunoactivity. The chromatogram isshown in FIG. 3.

EXAMPLE 4 Chromatography of IBP-2 and Preparation of IBP-5

Two hundred mg of IBP-2 were dissolved in 10 mL distilled water,pre-filtered with 0.45 μm filter, loaded on a Sephacryl S 300 HR column(2.5×90 cm), and eluted with 0.1 M acetate buffer, pH 4.5 and linearNaCl gradient. Chromatography was monitored at 280 nm, and at 490 nmafter interaction of the eluted fractions with-a phenol/sulfuric acidreagent. The mixture was resolved into two peaks. The majorimmunoactivity was in the high molecular mass peak (IBP-5). The lowermolecular mass peak (IBP-6) consisted of two fused peaks (an apparentpeak shoulder, the fractions of which were tested as IBP-6s, FIG. 5).The SEC profile is shown in FIG. 4.

EXAMPLE 5 Chromatography of IBP-2 and Preparation of IBP-7

Two hundred mg of IBP-2 were dissolved in 10 mL distilled water,pre-filtered with 0.45 μm filter, loaded on Sephadex G 100 column(2.5×90 cm), and eluted with 0.1 M acetate buffer, pH 4.5 and linearNaCl gradient. Chromatography was monitored at 280 nm, and at 490 nmafter interaction of the eluted fractions with a phenol/sulfuric acidreagent. The mixture was resolved into two peaks. The majorimmunoactivity was in the high molecular mass peak (IBP-7). The lowermolecular mass peak (IBP-8) retained much less of the activity. IBP-9represents the fractions following IBP-8 (FIG. 6).

EXAMPLE 6 Compositional Analysis

Dialysis or ultrafiltration demonstrated that only a small portion ofimmunoactivity was associated with low molecular mass (less than 100KDa) compounds. The active material is thermally stable and could beprecipitated from solution with ethanol or ammonium sulfate. The thermalstability of the extract makes it suitable for spray-drying; indeed,immunomodulatory activity was retained after the spray-drying process. Atypical protein content of a fraction is about 30%. DNA content variesfrom 0% to 20%, with 0% to 2% at molecular masses greater than 100 KDa.

(a) Carbohydrate Composition by Planar Chromatography and GasChromatograph (GC):

In one embodiment, the lyophilized extract (or further purifiedfractions) was dissolved in water (1 mg/mL, 400 μL) and hydrolysed with1M trifluoacetic acid (TFA, 1 mL) at 100° C., overnight with stirring,in tightly sealed 4 mL screw cap vials. The sample was then evaporatedrepeatedly to dryness using methanol. The dried hydrolysate was reducedusing 0.5 M NaBH₄ in 1 M NH₄OH (0.6 mL) under overnight stirring at roomtemperature. The borohydride was then quenched with acidic methanol (20%acetic acid in methanol, 1 mL) and the mixture evaporated to dryness.

Three mL of acetic anhydride were added to the sample; the mixture washeated in a water bath to 80° C. for 2 h to produce acetyl alditols andthen evaporated to dryness.

The acetyl alditol samples were extracted by distributing the reactionmixture between ethyl acetate and water; the organic phase was useddirectly for gas chromatography/mass spectroscopy (GC-MS) analysis.

All standards and sample extracts were dissolved in ethyl acetate andconcentrated to approximately 100 μL. Samples were injected using thesplit mode of a Thermoquest Trace 2000 gas chromatograph at a 10:1 splitand chromatographed on an SGE BPX70 capillary column (30 m×0.25 mm×0.25μm film thickness). Helium carrier gas was used at a constant flow rateof 1.0 mL/min.

The gas chromatograph oven was programmed at an initial temperature of190° C. (hold for 1 minute) followed by a 3° C./minute ramp to 260° C.(hold for 10 minutes at 260° C.). The capillary column was interfaceddirectly to the mass spectrometer (Thermoquest GCQ ion trap), with thetransfer line temperature at 260° C. Using this oven program, allcompounds of interest were found to elute within 20 minutes.

The mass spectrometer ion source was maintained at 150° C. Spectra wererecorded from m/z 50 to 500 using both electron impact mode (70 eV) andchemical ionization (CI) mode with ammonia reagent gas.

Retention times for the monosaccharides were established by derivatizingpure standards of individual sugars and/or chromatographing commerciallyavailable mixtures of alditol acetates (Supelco, Inc.). The sugarspresent in the sample extracts were identified by comparison ofretention times and mass spectra against these standards. All sampleswith immunomodulatory activity contain glucose, galactose, rhamnose,mannose and arabinose. Extracts of molecular weight greater than 1×10⁶Da are substantially free of ribose.

(b) Monosaccharide Compositional Analysis Using Polyacrylamide GelElectrophoresis (PAGE):

The standard hydrolysis protocols to release acidic, neutral or basicmonosaccharides from the polysaccharide backbones were employed. Theliberated monosaccharides were labeled with fluorescent labeling reagent2-aminoacridone (AMAC) first, followed by reduction of the formed Schiffbase with sodium cyanoborohydride. Polyacrylamide gel electrophoresis ofthe mixtures was then run on gradient gels according the instructionmanual. Hydrolysates produced from IBP-2 contained glucose, galactose,rhamnose mannose, and arabinose. A substantial mount of ribose was alsofound. However, the hydrolysates produced by treatment of IBP-5 andIBP-7 with 2M TFA at 100° C. for 5 h clearly contained only glucose andgalactose as well as rhamnose, mannose and arabinose. No ribose wasfound in these hydrolysates. This was in accord with our previousfindings that RNA fragments of low molecular mass were present in IBP-2.However, because of their small size they could not be present in highmolecular mass peaks of IBP-5 and IBP-7 obtained from Sephadex G 100 ofSephacryl S 300 chromatographies, respectively. Another band placedbetween N-acetyl galactosamine (GalNAc) and mannose was detected in PAGEbut could not be assigned to any of the conventional monosaccharides.The reaction mixtures obtained by hydrolysis performed under theconditions used for sialic acid release (0.1 TFA, 80° C., 1 h) resultedin a product with a Rf identical with that of sialic acid and anothermajor band with retention time significantly slower than thatcorresponding to GalNAc. Four molar HCl hydrolysis for 3 h at 100° C. (acondition for aminosugar release) resulted in PAGE in bandscorresponding to GalNAc and N-acetyl glucosamine (GlcNAc). Howeverjudging from the intensity of other bands they were insignificantcomponents of IBP-5 of IBP-7.

(c) Monosaccharide Compositional Analysis Using CapillaryElectrophoresis (CE):

The protocol followed was that of Sato et al. (Sato K., Okubo A.,Yamazaki T., (1997) Determination of monosaccharide derivatized with2-aminobenzoic acid by capillary electrophoresis. Anal Biochem 251:119-121). IBP-2 or its fractions were hydrolyzed first in 0.1 M TFA (1mg/ml), at 100° C. for 18 h, the aqueous acid was removed under reducedpressure, the residual TFA was removed by a sequential evaporation withmethanol until dryness.

The crude extract IBP-2 contains primarily glucose and galactose;glucose is the most prevalent monosaccharide in the extract. Alsopresent are rhamnose, mannose and arabinose, albeit in significantlysmaller quantities (FIG. 14). When the extract was subjected toultrafiltration using a 1 Mda MWCO membrane (FIG. 15), the ratio betweenglucose and galactose changed and the ribose peak disappeared,indicating (in agreement with PAGE data) removal of RNA and itsfragments.

(d) Amino Acid Composition:

The free amino acids contained in the extracts were determined asfollows: The samples were dissolved in distilled de-ionized water for afinal concentration of 10 mg/mL. To 25 μL (250 μg) of each sample, 75 μLof Beckman sample buffer (Na-S) was added. Samples were centrifuged at16,000×g to remove particulate matter before analysis on the BeckmanModel 6300 amino acid analyser. The centrifugation clarified thesolution and the pellet was retained; the supernatant was analysed forfree amino acids.

To determine the amino acid composition of whole hydrolysates, portionsof each sample (250 μg) were placed into a cleaned glass test tube alongwith the internal standard norleucine and 1 mL 6N HCl. The tubes weresealed under vacuum and the peptides and polypeptides hydrolysed at 105°C. for 20 hours. The tubes were opened and the samples were dried in aSavant Environmental Speed-vac at room temperature. The residues werere-dissolved in Na-S buffer and handled as described above for thedetermination of free amino acids.

(e) DNA Content:

The Chlorella extract was dissolved in distilled water (500 μL). Anequal volume of a phenol/chloroform/isoamyl alcohol (25:24:1, v/v)solution is added. The mixture is shaken vigorously then centrifuged at13,000 rpm for 5 minutes. The top aqueous layer is pipetted off andre-extracted with an additional 500 μL of the organic mixture. Theaqueous layer is re-extracted until the protein layer (a visibleinterface between the aqueous and organic layers) is negligible. Afterthe final protein extraction, the aqueous layer was transferred to afresh tube and to it was added glycogen (10 μL), 3M sodium acetate (50μL), and cold ethanol (1 mL). The mixture was shaken and placed in the−80° C. freezer for an hour. It was then centrifuged at 13,000 rpm for15 minutes and the supernatant was poured off. The pellet was dried onthe speed-vacuuming and re-dissolved in water. Its absorbance wasmeasured at 260 nm against a water blank. Since proteins also absorb at260 nm, protein absorbance readings at 280 nm were also taken as ameasure of protein content in the DNA sample.

EXAMPLE 7 Enzyme Digestion of Extracts

Enzyme degradation was used as a technique to selectively eliminatevarious macromolecular classes from the Chlorella extracts. IBP-2 or itsfractions (both IBP-2 and its fractions are designated ONC-107 in thisexample) were treated with pronase, DNAse, RNAse, amylase,amyloglucosidase, cellulase and neuraminidase in separate experiments.SDS page and agarose electrophoresis as well as thin layerchromatography were used to monitor the enzymatic reactions (seesections (a) to (g) below). The final reaction mixtures were dialyzed,lyophilized and tested for the capacity to stimulate undifferentiatedspleen cells by thymidine incorporation into murine splenocytes(Examples 9 and 11). The results are summarized as follows:

-   -   No effect on the capacity to stimulate undifferentiated spleen        cells within a statistical significance after treatment with the        selected enzymes.    -   No presence of large molecular mass RNA in IBP-2. This is in        agreement with other findings (above), specifically that no        ribose was found in high molecular mass species (IBP-5, IBP-7)        and in fractions ultrafiltered with MWCO>1 MDa. This clearly        demonstrates that RNA is not the primary source of the        immunostimulating activity.    -   A very small quantity of DNA was found, which is in accord with        our data obtained from the isolation of DNA using a silicon        carbide column (about 2% content, 500 bp) (see Haj-Ahmad        Y (1999) Nucleic acid purification and process. Canadian        published application 2,270,106).

All enzyme digestions were performed in parallel with positive controlsto ensure that the enzymes were active. The experiments clearly indicatethat unassociated, enzyme-accessible protein and nucleic acids areunlikely to be sources of the activity. The source of immunoactivity isa polysaccharide (perhaps complexed with another biopolymer which has nosignificant effect by itself and which might have an indirect role, e.g.stability). The immunoactivity of the polysaccharide is not affected bycleavages in the regions of three or more α-1,4-linked D-glucose units,α-1,4-linked glucosides and galactosides, or α-1,4-linked D-glucose,assuming such linkages are accessible to enzyme cleavage.

(a) Protease Treatment

Protease (100 μg/ml, Streptomyces griseus) was added to ONC-107 (20mg/ml) in TRIS buffer (0.05M, pH 7.4) and incubated for 1 hour at 36° C.Incubation was stopped by heat deactivation of the enzyme at 80° C. for1 hour followed by centrifugation for 10 minutes at 13000 rpm. Aliquotswere taken from the solution at time intervals of 0, 5, 10, 15, 30 and60 minutes and prepared for analysis by SDS-PAGE electrophoresis (12%gel, total protein stain). The optimum concentration of protease wasdetermined by a similar electrophoretic time course involving BSA (1mg/ml). The final digest mixture was analyzed by agarose electrophoresis(1% stained with ethidium bromide).

(b) DNAse Treatment

DNAse (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRIS buffer (0.05M,pH 7.4, 10 mM MgCl₂) and incubated for 1 hour at 36° C. Incubation wasstopped by heat deactivation of the enzyme at 80° C. for 1 hour followedby centrifugation for 10 minutes at 13000 rpm. The final digest mixturewas analyzed by SDS-PAGE electrophoresis (12% gel, total protein stain)to check for protein degradation. Agarose electrophoresis (1% stainedwith ethidium bromide) was used to confirm nucleic acid degradation.

(c) RNAse Treatment

RNAse (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRIS buffer (0.05M,pH 7.4, 10 mM NaCl) and incubated for 1 hour at 36° C. Incubation wasstopped by heat deactivation of the enzyme at 80° C. for 1 hour followedby centrifugation for 10 minutes at 13000 rpm. The final digest mixturewas analyzed by SDS-PAGE electrophoresis (12% gel, total protein stain)to check for protein degradation. Agarose electrophoresis (1% stainedwith ethidium bromide) was used to confirm nucleic acid degradation.

(d) Amylase Treatment

Amylase (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRIS buffer(0.05M, pH 7.4) and incubated for 1 hour at 36° C. Incubation wasstopped by heat deactivation of the enzyme at 80° C. for 1 hour followedby centrifugation for 10 minutes at 13000 rpm. The final digest mixturewas analyzed by SDS-PAGE electrophoresis (12% gel, total protein stain)to check for protein degradation. Agarose electrophoresis (1% stainedwith ethidium bromide) was used to confirm nucleic acid degradation.

Additionally, thin layer chromatography (TLC) confirmed the activity ofthe enzyme as follows. Amylase (100 u g/ml) was added to a solution ofstarch (1 mg/ml) and incubated for one hour at 36° C. The solution wasanalyzed via TLC using keisgel silica plates eluted withisopropanol:ethylacetate:water (7:1:2). The plates were developed after10 minutes drying in the horizontal position using a sulfuricacid/ethanol solution. Glucose (1 mg/ml) was used as a control as wasuntreated starch. Treatment of starch with amylase resulted in theliberation of glucose.

(e) Amyloglucosidase

Amyloglucosidase (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRISbuffer (0.05M, pH 4.4) and incubated for 1 hour at 36° C. Incubation wasstopped by heat deactivation of the enzyme at 80° C. for 1 hour followedby centrifugation for 10 minutes at 13000 rpm. TLC confirmed theactivity of the enzyme as follows. Amyloglucosidase (100 ug/ml) wasadded to a solution of starch (1 mg/ml) and incubated for one hour at36° C. The solution was analyzed via TLC using keisgel silica plateseluted with isopropanol:ethylacetate:water (7:1:2). The plates weredeveloped after 10 minutes drying in the horizontal position using asulfuric acid/ethanol solution. Glucose (1 mg/ml) was used as a controlas was untreated starch. Treatment of starch with the amyloglucosidaseresulted in the liberation of glucose.

(f) Cellulase

Cellulase (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRIS buffer(0.05M, pH 7.4) and incubated for 1 hour at 36° C. Incubation wasstopped by heat deactivation of the enzyme at 80° C. for 1 hour followedby centrifugation for 10 minutes at 13000 rpm. TLC confirmed theactivity of the enzyme as follows. Cellulase (100 ug/ml) was added to asolution of cellulose (1 mg/ml) and incubated for one hour at 36° C. Thesolution was analyzed via TLC using keisgel silica plates eluted withisopropanol:ethylacetate:water (7:1:2). The plates were developed after10 minutes drying in the horizontal position using a sulfuricacid/ethanol solution. Glucose (1 mg/ml) was used as a control as wasuntreated cellulose. Treatment of cellulose with cellulase resulted inthe liberation of glucose.

(g) Neurominidase

Neurominidase (100 μg/ml) was added to ONC-107 (20 mg/ml) in TRIS buffer(0.05M, pH 5.0) and incubated for 1 hour at 36° C. Incubation wasstopped by heat deactivation of the enzyme at 80° C. for 1 hour followedby centrifugation for 10 minutes at 13000 rpm. TLC confirmed theactivity of the enzyme as follows. Neurominidase (100 ug/ml) was addedto a solution of N-acetyl-neuromidose (1 mg/ml) and incubated for onehour at 36° C. The solution was analyzed via TLC using keisgel silicaplates eluted with isopropanol:ethylacetate:water (7:1:2). The plateswere developed after 10 minutes drying in the horizontal position usinga sulfuric acid/ethanol solution.

EXAMPLE 8 Stimulation of Splenocyte Proliferation

Fresh splenocyte cells (splenocyte primary culture) were plated at 3×10⁵cells/well in cRPMI medium in a 100 μl volume in 96-well flat bottomtissue culture plates. Test samples in 100 μl of cell medium were addedto triplicate wells giving a final total volume in each well of 200 μl.The plates were covered with sterile lids and incubated in 5% CO₂ at 37°C. and 100% humidity in a CO₂ incubator for 48 h. The cells were thenpulsed with ³H-thymidine (1 μCi per well in 10 μl cRPMI) and incubatedfor 18 h. Cells were harvested with an automated cell harvester equippedwith filter strips. The filter strips were allowed to dry for 3 h at 37°C. The radioactivity incorporated by the cells was determined bycounting the filter strips immersed in scintillation medium in a liquidscintillation counter. An example of the experiment is illustrated inFIG. 7. A comparison of applicant's extract with two commercial samplesis illustrated in FIG. 13.

EXAMPLE 9 Isolation of B-Cells and T-Cell from Splenocytes andStimulation of the Cells

Mouse splenocytes were isolated by routine methods and placed in tissueculture flasks at 37° C. for 2 hr, to allow the macrophages to settleand adhere to the flask. The lymphocytes (which are suspended and do notadhere) are then removed and placed in a 50 ml centrifuge tube, spun andresuspended in a small amount of cold PBS-EDTA-BSA buffer (pH 7.4; 1-3ml) and placed on ice. Cells were counted and recentrifuged. They werethen resuspended at 1×10⁸ in 0.3 ml of the PBS-EDTA-BSA buffer in a 15ml centrifuge tube.

Negative isolation of B-cells: 100 μl of Miltenyi microbeads coated withanti-Thy-1.2 antibody was added to the resuspended cells, and themixture was incubated for 20 minutes. PBS-EDTA-BSA (5 mL) was added andthe suspension was pipetted into a midiMACS column in a midiMACS magnet.The column had a 25-gauge needle on the outflow to restrict the flowrate. The T-cells, bound to the anti-Thy-1.2 antibody-coated magneticbeads, adhered to the column. The B-cells passed through and werecollected. The column, without being removed from the magnet, was rinsedwith 5 ml of the buffer to remove any residual B-cells. The B-cells werecombined, spun, resuspended in 1 ml of cRPMI and counted. They were thenresuspended to 5×10⁶ cells per ml of cRPMI and plated at 100 μl/well forstimulation assays.

Negative isolation of T-cells: The same procedure was followed as forisolation of B-cells, except the magnetic beads used for the incubationwere coated with anti-B220 antibody instead of anti-Thy-1.2.

The purified T- and B-cell populations were tested as described inExample 8. The results are shown in FIG. 8.

EXAMPLE 10 Nitrite Assay for Mouse Peritoneal Macrophages

Mice were euthanized by cervical dislocation, and placed spreadeagled ontheir backs. Their abdomens were sterilized with 70% alcohol, and acareful midline incision exposing the INTACT peritoneal wall was made. A10-ml syringe was used to inject 10 ml of cold cRPMI-1640 into the mouseperitoneal cavity. The mice were gently rocked from side to side withthe needle still inserted. The cRPMI-1640 containing the peritonealmacrophages was slowly drawn from the peritoneal cavity through theneedle. About 8 ml of fluid per mouse was recovered. The peritonealfluid was pooled and put into 50 mL centrifuge tubes on ice. The cellswere spun down, washed and resuspended in 1 ml of cRPMI-1640. Aftercounting, they were resuspended to 1-2×10⁶ cells/ml and plated into a96-well tissue culture plate at 1×10⁵ cells/well in a 100 μl volume.Test samples were added in cRPMI (100 μl) with and without IFN-μ. Thepositive controls were the cells +IFN-μ and LPS+IFN-μ. The cells wereincubated for 48 h in a CO₂ incubator in 5% CO₂.

Fifty μl of the culture fluid was collected and transferred to wells ina 96-well flat bottom ELISA plate. Twofold serial dilutions of NaNO₂(125 μM to 1 μM final concentration) in cRPMI were made, and 50 μl ofeach Greiss Reagent solution and the NaNO₂ dilutions were added to a setof wells to provide a standard curve. Absorbance at 550 nm was measuredand a plot of absorbance values against NaNO₂ concentrations was made.The standard curve was used to determine the amount of NO₂ ⁻ produced bythe peritoneal macrophage samples. An example of the experiment isillustrated in FIG. 9.

EXAMPLE 11 Determination by Sandwich ELISA of Stimulation of CytokineProduction by Mouse Splenocytes

Cytokine production was measured in mouse splenocytes, in separatedmouse T- and B-lymphocytes, and in mouse macrophages. The test sampleswere added at several concentrations to the cells in microtiter tissueculture plates. After incubating for 24-48 h, depending on the cytokineof interest, the supernatant culture fluid was removed for ELISA.

ELISA plates were coated with anti-cytokine monoclonal antibodies byincubation at 4° C. overnight in a carbonate buffer, pH 9.6. The plateswere then washed with Tris-buffered saline (TBS), post-coated with 2mg/ml BSA in TBS, (200 μl/well) for 2 h at room temperature and washedwith TBS/Tween. The samples and standards (the latter diluted 1 ng/ml to15 pg/ml in twofold dilutions) were diluted in TBS/Tween containing 1mg/ml BSA (100 μl/well), added to the plate, incubated overnight at 4°C. and then washed with TBS-Tween.

The appropriate biotinylated anti-cytokine mAb (0.5 μg/ml) in PBS-Tweencontaining 1 mg/ml BSA (100 μl/well), was added. The plate was incubatedat room temperature for 1 h and then washed with TBS-Tween.Extravidin-Peroxidase in PBS-Tween containing 1 mg/ml BSA (100 μl/well)was added and incubated at room temperature for 30 minutes. The plateswere then washed. One hundred μl/well of TMB substrate solution wasadded, and after 10 to 30 minutes, depending on color development, thereaction was stopped with 100 μl/well of 1 M H₃PO₄. The plate was readat 450 nm. An example of stimulation of IL-6 production is illustratedin FIG. 10.

EXAMPLE 12 Effect of IBP-2 on Proliferation of Listeria monocytogenes inInfected Mice

Two doses of IBP-2 (0.1 mg or 4 mg) or plain water (negative control)were administered to Balb/c mice by intragastric tube three times a weekfor four weeks. The mice were then infected by intravenous injection of5,000 viable Listeria monocytogenes organisms. Three days after theListeria injections, the mice were sacrificed. Cell suspensions of theirspleens were made and cultured on culture dishes to determine the numberof bacteria in the spleens. The water-fed animals had 92,202 (±23,000)bacterial colony forming units (CFUs) in their spleens; (the number inbracket refers to the standard deviation). The animals fed 0.1 mg ofIBP-2 per dose had 43,310 (±7,021) CFUs and the animals fed 4 mg ofIBP-2 per dose had only 5,317 (±492) CFUs (p<0.05) (FIG. 11).

EXAMPLE 13 Effect of IBP-2 on Proliferation of Candida albicans inInfected Mice

IBP-2 (4 mg), crude algae (4 mg or 20 mg), or plain water wasadministered to Balb/c mice by intragastric tube 3 times per week fortwo weeks. The mice were then infected by intravenous injection of500,000 viable Candida albicans organisms. Feeding continued until themice were sacrificed 12 days after infection. The kidneys were removedand cell suspensions made and cultured on Sabouraud agar to determinethe number of C. albicans colonies which developed overnight. Thefollowing results were obtained: water fed mice 594 (±556) colonies(mean±standard deviation); IBP-2 (4 mg) fed mice 42 (±75) (p<0.05compared to water fed mice); algae (4 mg) fed mice 335 (±663); algae (20mg) fed mice 79 (±70); (statistically, the p value for this groupcompared to the water fed group was >0.05, although it was very close tobeing significant). The results are illustrated in FIG. 12.

EXAMPLE 14 Phase 2 Clinical Trial Study

This study was designed to assess the immunostimulatory efficacy ofChlorella extracts as a nutritional supplement in healthy adults over 50years of age. A first-in-man study was completed which demonstrated thesafety and tolerability of a Chlorella extract corresponding to IBP-1when given as a once daily supplement over three weeks. This purpose ofthis phase 2 study was to increase the human experience with Chlorellaextracts through increased safety and tolerability assessment and toexplore the capacity of Chlorella extracts as an immune stimulatingnutritional supplement in humans.

The study was designed as a randomized, placebo controlled clinicaltrial in which adults 50 years of age or older were assigned by chanceto receive a 200 mg capsule of a Chlorella extract corresponding toIBP-1, (designated ONC-107 for the purpose of this study), a 400 mgcapsule of ONC-107, or a placebo capsule (containing no ONC-107). Whilethe trial was underway, the investigators, the nurses and other studypersonnel and the participants were not aware to which group they wereassigned. The safety and side effects were measured by careful recordingby the participant of any adverse event and reporting these to studypersonnel. Specific adverse events measured included fever, abdominalpain, nausea, vomiting, diarrhea, fatigue, decreased appetite, headache,body aches, sore joints, and rash. Safety was also measured by a seriesof blood tests before starting the study capsules and at the completionof the study capsules. These tests included tests of liver function(AST, ALT), blood profiles (complete blood count), and immunologicalfunction (ANA, anti-DNA, rheumatoid factor, Coombs, C3, C4, quantitativeIgG, IgA, IgM, and IgE). Medication was taken for 28 days; after 21days, participants were immunized with a commercially available,inactivated, split virion influenza vaccine. Antibody response to theimmunization was assessed by measuring antibodies to the three influenzavirus strains before, and 7 and 21 days after immunization. Cellmediated immunity was measured by evaluating the response to aninfluenza skin test at the beginning of the study and one week after theimmunization.

A total of 124 subjects were enrolled into the study and took the studymedication; 7 participants withdrew from the study. Only one participantwithdrew because of adverse events (nausea and abdominal pain). Thethree treatment groups were similar in age, gender and baseline historyand physical examination at the commencement of the study. The majorityof participants were women (73.2-80.5% of each group). Participantcompliance was excellent. Antibody response to the influenza vaccine wasnot significantly higher in the ONC-107 treated participants overallalthough in participants 55 years of age or younger there was asignificantly enhanced response to some antibody measures (and aconsistent trend to the others). There were no serious adverse events inany of the study participants. An adverse event was reported by mostparticipants at sometime during the study but, for the most part, theseevents were not reported more frequently in the ONC-107 recipientscompared to the placebo recipients (fatigue was reported more frequentlyby 200 mg ONC-107 recipients and abdominal pain more frequently byplacebo recipients older than 55 years of age). There were nosignificant changes in laboratory measurements before and after therapy,or between recipients of the ONC-107 and placebo.

The results of this phase 2 study of the nutritional supplement ONC-107at doses of 200 mg and 400 mg for 28 days in healthy adults are outlinedbelow. They indicate that, despite an age effect, this product is welltolerated and safe for oral administration and has a measurableimmunostimulatory effect.

The immunostimulatory effect was measured by antibody response toinfluenza vaccine in healthy adults over 50 years of age, although theseresponses were in general limited to the younger cohort in the studypopulation (50-55 years of age). The increase in antibody response inthis subgroup was statistically significant for some of the comparisonsbut the trend was apparent in all serological comparisons made. Thepre-study hypothesis that the effect of ONC-107 would be best observedin the older subjects because of their decreased responsiveness toinfluenza vaccine was not supported by the data; in contrast, it was theyounger, more responsive subjects who demonstrated an effect of theONC-107.

The younger subjects tended to show immunostimulatory effects of ONC-107(especially 400 mg dose). A potential reason for the lack of effect inthe older age group was that the older group may have had higherpre-immunization antibody titers which may indicate a greater degree ofprior exposure to antigenically similar flu virus A strains (but not theB strains).

1. For the A/Caledonia strain of the flu vaccine, the younger (<=55yrs.) age group had higher mean antibody titers with both doses thanplacebo at both 7 (not significant at 7 d) and 21 days (p=0.05):placebo=43.2; 200 mg=84.3; 400 mg=84.4.

2. For the B/Yamanashi strain of the flu vaccine, the 400 mg group hadhigher titers (30.1) at 7 days vs. placebo (14.4, p=0.03), but this wasnot significant at 21 days. The 200 mg dose was not significantlygreater than placebo at 7 days (15.9) or 21 days (25.3).

3. For A/Panama strain of the flu vaccine, there was a similar trend infavour of 400 mg dose, but it was not significant. At 7 days, the 400 mggroup had titers of 64.5, compared to placebo (39.9), which is notsignificant. The 200 mg group has titers of 26.6. At 21 days, there wasalso a trend in favour of 400 mg vs. placebo; again, 200 mg was notbetter than placebo (57.4).

Similar trends were observed for the proportion of subjects having 2-and 4-fold antibody responses.

For the B/Yamanashi in the <55 yr. group, only 5% of the placebo had a2-fold antibody response at 7 days. This is in contrast with the 400 mggroup (41.2%, p=0.01) and 200 mg group (6.3%, p=0.04).

There was also a non-significant trend for an increased response at 21days, and a non-significant trend in the proportion of the <55 yr. agegroup achieving seroprotective levels of 40 RD (reciprocal dilutions) at7 days post-immunization.

Numerous modifications, variations and adaptations may be made to theparticular embodiments of the invention described above withoutdeparting from the scope of the invention which is defined in theclaims.

1. A process for obtaining a Chlorella composition comprising highmolecular weight polysaccharide and polysaccharide complexes, thecomposition having immunomodulating activity, wherein the processcomprises the steps of: (a) size fractionating an aqueous extract ofChlorella, and (b) selecting fractions comprising high molecular weightpolysaccharide and polysaccharide complexes of about 3×10⁵ Da to about1×10⁷ Da, such that the high molecular weight polysaccharide andpolysaccharide complexes of about 3×10⁵ Da to about 1×10⁷ Da aboutconstitute at least 22% (w/w) of total Chlorella-derived content of theextract.
 2. The process of claim 1 further comprising the step ofpooling and concentrating the selected fractions.
 3. A Chlorellacomposition comprising high molecular weight polysaccharide andpolysacoharide complexes of about 3×10⁵ Da to about 1×10⁷ Da obtainedfrom the process of claim
 2. 4. The process of claim 1 wherein the sizefractionating step comprises chromatography or ultrafiltration.
 5. AChlorella composition comprising high molecular weight polysaccharideand polysaccharide complexes of about 3×10⁵ Da to about 1×10⁷ Da,obtained from the process of claim
 1. 6. The process of claim 1 whereinthe fractions selected in step (b) comprise high molecular weightpolysaccharide and polysaccharide complexes of about 3×10⁵ Da to about3×10⁶ Da.
 7. A Chlorella composition comprising high molecular weightpolysaccharide and polysaccharide complexes of about 3×10⁵ Da to about3×10⁶ Da obtained from the process of claim
 6. 8. The process of claim 1wherein the fractions selected in step (b) comprise high molecularweight polysaccharide and polysaccharide complexes of about 3×10⁶ Da toabout 7×10⁶ Da.
 9. A Chlorella composition comprising high molecularweight polysaccharide and polysaccharide complexes of about 3×10⁶ Da toabout 7×10⁶ Da obtained from the process of claim
 8. 10. The process ofclaim 1 wherein the fractions selected in step (b) comprise highmolecular weight polysaccharide and polysaccharide complexes of about7×10⁶ Da to about 1×10⁷ Da.
 11. A Chlorella composition comprising highmolecular weight polysaccharide and polysaccharide complexes of about7×10⁶ Da to about 1×10⁷ Da obtained from the process of claim
 10. 12. Amethod for modulating an immune response of a mammal, the methodcomprising administering to the mammal an effective amount of an extractcomprising high molecular weight Chlorella polysaccharide andpolysaccharide complexes, wherein the high molecular weightpolysaccharide and polysaccharide complexes are about 3×10⁵ Da to about1×10⁷ Da and constitute at least 22% (w/w) of total Chlorella-derivedcontent of the extract.
 13. The method of claim 12 wherein modulationcomprises increased proliferation of splenocytes.
 14. The method ofclaim 12 wherein modulation comprises increased production of acytokine.
 15. The method of claim 14 wherein the cytokine is selectedfrom the group consisting of IL-6, IL-10, INF-γ and TNF-α.
 16. A methodfor supplementing an immune response to a vaccine in a mammal, themethod comprising administering to the mammal being vaccinated aneffective amount of an extract comprising high molecular weightChlorella polysaccharide and polysaccharide complexes, wherein the highmolecular weight polysaccharide and polysaccharide complexes are about3×10⁵ Da to about 1×10⁷ Da and constitute at least 22% (w/w) of totalChlorella-derived content of the extract.
 17. The method of claim 16wherein the vaccine is a flu vaccine.
 18. A method for preventing ortreating bacterial infection in a mammal, the method comprisingadministering to the mammal subject to infection an effective amount ofan extract comprising high molecular weight Chlorella polysaccharide andpolysaccharide complexes, wherein the high molecular weightpolysaccharide and polysaccharide complexes are about 3×10⁵ Da to about1×10⁷ Da and constitute at least 22% (w/w) of total Chlorella-derivedcontent of the extract.
 19. A method for preventing or treating fungalinfection in a mammal, the method comprising administering to the mammalsubject to infection an effective amount of an extract comprising highmolecular weight Chlorella polysaccharide and polysaccharide complexes,wherein the high molecular weight polysaccharide and polysaccharidecomplexes are about 3×10⁵ Da to about 1×10⁷ Da and constitute at least22% (w/w) of total Chlorella-derived content of the extract.
 20. Amethod for vaccinating a mammal, the method comprising administering tothe mammal a vaccine and an effective amount of an extract comprisinghigh molecular weight Chlorella polysaccharide and polysaccharidecomplexes, wherein the high molecular weight polysaccharide andpolysaccharide complexes are about 3×10⁵ Da to about 1×10⁷ Da andconstitute at least 22% (w/w) of total Chlorella-derived content of theextract, wherein the vaccine and extract are administered together orseparately.